We would like to understand the genetic basis of amyotrophic lateral sclerosis (ALS). For the familial form of the disease, mutations in SOD1 are implicated in pathogenesis but account for only a minority of cases (approx 15-20%). It is our goal to identify new genes that contribute to the etiology of ALS. We recently responded to an RFA from NIH with this goal (NIMDS-RFA-NS-01-004). We propose a novel, forward genetic screen in mice treated with ethly-nitroso-urea for mutants showing signs of ALS. This proposal is part of a large collaborative centre recently funded by the Canadian Foundation for Innovation and the MRC genome program which is now generating 5000 ENU mice per year. No one in the USA or elsewhere is carrying out such a screen. Our goal in this R21 is to generate pilot data that will show the feasibility of a screen for the clinical signs of ALS. This will include changes in gait and stride, loss of limb function and paralysis. Offspring of those mutants will be screened for neuropatholgy that includes a loss of motor neurons and muscle denervation. We will count Chat+ and Nissl+ cells in the facial nucleus and level T12-L3 of the spinal cord. Axon counts will be made on the facial nerve and the L4 ventral roots. A loss in cell number should be paralleled by an increase in Caspase+ve, Tunnel +ve cells. We will also measure disease onset. Mutants with both a decline in motor function and a loss of motorneurons will be selected for further study. Mutant mice will be crossed to C3H/HeJ for rough mapping to a chromosomal position by the core. We will focus on novel genes that do not map to the Sod 1 locus. In future years, we will carry out fine mapping to 0.1 cm and BAC contigs spanning this region will be generated. Candidates will be confirmed by transgenic rescue of ALS mutant mice by gene replacement with the mutated gene. Candidate genes will be sequenced and screened for loss of mRNA in affected tissue: motor cortex and spinal-motor neurons. In future years additional mutant alleles will be cloned and eventually sequenced. We will isolate suppressors and enhancers and elaborate the entire genetic pathway leading to ALS in mice. We will ultimately establish if the mechanism of gene action that leads to motorneuron loss involves oxygen radical toxicity, glutamate excitotoxicity, loss of trophic support or novel pathways